P594: THE CXCR4-LOW/CD5-HIGH PROLIFERATIVE FRACTION IS ENRICHED IN BTK MUTATIONS AND ANTICIPATES RELAPSE IN IBRUTINIB-TREATED CLL
Authors: Federico Pozzo, Gabriela Forestieri, Giulia Ianna, Filippo Vit, Erika Tissino, Tamara Bittolo, Robel Papotti, Lodovico Terzi DI Bergamo, Agostino Steffan, Roberta Laureana, Agostino Tafuri, Annalisa Chiarenza, Francesco DI Raimondo, Jacopo Olivieri, Francesco Zaja, Luca Laurenti, Maria Ilaria DEL Principe, Riccardo Bomben, Antonella Zucchetto, Davide Rossi, Valter Gattei
Journal: HemaSphere (2023)

Background: CXCR4/CD5 expression on circulating chronic lymphocytic leukemia (CLL) cells discriminate the proliferative fraction (CD5high/CXCR4dim, PF), recently egressed from the lymph node, from the resting fraction (CXCR4high/CD5dim, RF) of older quiescent cells. BTK inhibitors affect the proliferative capacity of CLL cells and cause their redistribution from lymph nodes to peripheral blood (PB). Aims: In long-term Ibrutinib (IB)-treated CLL, to investigate the evolution of PF and correlate with clinical outcome. Methods: PB samples were from CLL patients entering NCT02827617 trial (n=31) or referred for routine (immuno)genetic analyses (n=100). Methods employed include: flow cytometry for PF/RF analyses (Fig.A), reported as median %, DNA and RNA-seq (Illumina) analyzed with R package DESeq2. Results: Longitudinal analysis of the PF under IB from trial CLL (156 sequential samples, median 6/case) showed depletion of the PF, from 17.7% at pre-treatment to 4.75-0.4-1.5-0.6-1.5% at 0.5-6-12-18-24 months (p < 0.001) with loss of Ki67 expression. By analyzing the PF from IB-treated real-world CLL (296 samples, median 3/case; Fig.A-B), median PF was 12.0% (range 0.7-50) in pre-IB samples with a drop to 2.7% at 1 year of treatment and 1.0% in the second year (p<0.001). IB was discontinued within 5 years in 71/100 cases (17 toxicity, 46 progression, 8 other/death). BTK/PLCG2 mutations were detected in 29/100 cases; among these, 24 discontinued IB due to progression/relapse (23/24) or toxicity (1/24). Focusing on samples collected within 12 months before/after IB discontinuation (n=86), mutations were represented in progressing cases (22/43, 51%) with a PF higher than wild-type (WT) cases discontinuing for toxicity (8.9% vs. 2.4%; p=0.012) or still on treatment (2.8%, p=0.015; Fig.C). Notably, the 21 WT progressing cases also showed higher PF compared to cases discontinuing for toxicity (6.9% vs. 2.4%; p=0.004) or still on treatment (6.9% vs. 2.8%; p=0.024). Sequential dosages of plasmatic B2M in progressing cases (n=5) revealed steady levels (median 2.38 ug/mL) below the 3.5 ug/mL threshold, despite the PF increase (median PF 18%; Fig.D). The PF/RF fractions were sorted from 10 cases developing BTK mutations with PF reappearance after prolonged IB treatment (median 50.7 months). Sequencing revealed 27 BTK mutations (mean 1.7/sample, range 1-4), with median VAF higher in the PF (23.0%, 0.3-92.3) than in the RF (5.2%, 0.1-84.8; p=0.003); overall, the PF VAF was 2.8-fold larger than the RF (0.01-38). A similar trend was present for PLCG2 mutations (median PF VAF: 2.0%, 0.1-6.2; RF VAF: 1.35%, 0.4-2.8; p=0.014; Fig.E). Finally, mRNA-seq was performed on separate PF/RF fractions from samples (n=6) at pre-IB and progression, the latter enriched in BTK mutations. Differential expression signature of pre-IB PF vs. RF, concordant with published gene sets, drove co-clustering of post-IB PF/RF with their respective counterparts (Fig.F), indicating that PF at progression functionally resembles its pre-treatment counterpart. Summary/Conclusion: Here we report: i) that IB treatment abrogates the PF fraction within 1 year of treatment, expanding previous short-term observations; ii) its reconstitution after long-term treatment may predict clinical progression anticipating B2M increase; iii) the enrichment of BTK/PLCG2 mutations in the reconstituted PF suggests the presence of active clonal selection dynamics, bona fide taking place within the lymph node microenvironment. Clinically, longitudinal monitoring of the CXCR4/CD5 fractions by flow cytometry may provide a simple tool helping to intercept CLL progression under IB therapy.Keywords: B-CLL, Relapse, ibrutinib, Prognostic factor